欢迎您, 登录 | 注册

首页 | English

X
加载中

Methyltransferase (MTase) transfers a methyl group (-CH3) from the donor S-adenosyl-L-methionine (AdoMet or SAM) to biologically active molecules such as hormones, neurotransmitters, lipids, proteins and nucleic acids. The addition of a methyl group causes a change in the physicochemical properties of the molecules. The mRNA cap structure is essential for cell and virus. Guanine-N7-methyltransferase (N7-MTase) methylates the GpppN cap at the N7 position of guanine, resulting in cap-0 structure (m7GpppN), and Ribose 2'-O-MTase further methylates the first nucleotide of higher eukaryotic cellular and viral mRNAs at the ribose 2'-OH position to form cap-1 (m7GpppNm) structures. Here, we describe a biochemical assay to detect the activities of mRNA capping MTases.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Biochemical Assays for MTase Activity
生物化学分析法检测甲基转移酶活性

微生物学 > 微生物生物化学 > 蛋白质 > 活性
作者: Yu Chen
Yu ChenAffiliation: College of Life Sciences, Wuhan University, Wuhan, China
For correspondence: chenyu@whu.edu.cn
Bio-protocol author page: a1107
 and Deyin Guo
Deyin GuoAffiliation: College of Life Sciences, Wuhan University, Wuhan, China
For correspondence: dguo@whu.edu.cn
Bio-protocol author page: a1108
Vol 4, Iss 2, 1/20/2014, 1815 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.1023

[Abstract] Methyltransferase (MTase) transfers a methyl group (-CH3) from the donor S-adenosyl-L-methionine (AdoMet or SAM) to biologically active molecules such as hormones, neurotransmitters, lipids, proteins and nucleic acids. The addition of a methyl group causes a change in the physicochemical properties of the molecules. The mRNA cap structure is essential for cell and virus. Guanine-N7-methyltransferase (N7-MTase) methylates the GpppN cap at the N7 position of guanine, resulting in cap-0 structure (m7GpppN), and Ribose 2'-O-MTase further methylates the first nucleotide of higher eukaryotic cellular and viral mRNAs at the ribose 2'-OH position to form cap-1 (m7GpppNm) structures. Here, we describe a biochemical assay to detect the activities of mRNA capping MTases.

Keywords: Methyltransferase(甲基转移酶), S-adenosyl-L-methionine(S-腺苷甲硫氨酸), RNA capping(RNA封盖), Cap structure(盖结构)

Materials and Reagents

  1. Bodicon m7G capping system (Bodicon, catalog number: CS0130)
  2. S-adenosyl methionine (SAM) (involved in Bodicon m7G capping system) (Bodicon, catalog number: CS0130)
  3. Bodicon Capping Enzyme (10 U/µl) (involved in Bodicon m7G capping system) (Bodicon, catalog number: CS0130)
    Note: Because the sale of this kit was low, the previous companies which provided this capping kit were out of service. This capping kit was provided by a new company in China as custom-made products (contact e-mail: service@bodicon.cn, phone:+86-13628662011). In fact the similar capping kit from any other companies (such as EPICENTRE biotechnologies, ScriptCap m7G capping system, catalog number: SCCE0610) is suitable for this experiment, and people can also contact with us to get the related protein or kit.
  4. Inorganic pyrophospatase (YIPP) (New England Biolabs, catalog number: M2403S)
  5. S-adenosyl [methyl-3H] methionine ([3H]-SAM) (PerkinElmer, catalog number: NET155H001MC)
  6. DEAE Sephadex (GE Healthcare, catalog number: 17-0170-01)
  7. GTP (Thomas Scientific, catalog number: R0461)
  8. RNase inhibitor (Thomas Scientific, catalog number: EO0381)
  9. RNase free water
  10. Phenol-chloroform (pH 4.8-5.2 for RNA only)
  11. Ethanol (RNase free)
  12. RNase free water
  13. Sodium Dodecyl Sulfonate (SDS)
  14. Ethylene Diamine Tetraacetic Acid (EDTA)
  15. NH4HCO3
  16. NaCl
  17. 10x MTase Buffer (see Recipes)
  18. Cap-0 cap structure (m7GpppN-RNA) (see Recipes)
  19. Non-methylated Cap-0 cap structure (GpppN-RNA) (see Recipes)
  20. MTase assay reaction mix (see Recipes)

Equipment

  1. Bechtop
  2. Water bath
  3. Centrifuge
  4. Liquid scintillation counter

Procedure

  1. Synthesis of RNA cap structure as substrates.
    The nascent RNA transcribed in vitro possesses a 5' triphosphate end. Two RNA capping systems are used to synthesize cap structure as the substrates of MTase.
    1. Combine 1-10 µg RNA and RNase free water up to 12.5 µl of total reaction volume.
    2. To heat to denature the in vitro transcribed RNA, incubate the tube at 65 °C for 10 min, and transfer the tube to ice immediately.
    3. Combine the following reaction components in the order given:
      Cap-0 cap structure (m7GpppN-RNA)
      Heat denatured RNA   
      12.5 µl
      10x Bodicon Capping Buffer   
      2 µl
      10 mM GTP   
      2 µl
      3 mM SAM   
      1 µl
      RNase inhibitor 40 U/µl   
      0.5 µl
      Bodicon Capping Enzyme (10 U/µl)   
      2 µl
      Total   
      20 µl
      Non-methylated Cap-0 cap structure (GpppN-RNA)
      Heat denatured RNA   
      12.5 µl
      10x Bodicon Capping Buffer   
      2 µl
      10 mM GTP   
      2 µl
      Inorganic pyrophospatase 0.1 U/µl   
      1 µl
      RNase inhibitor 40 U/µl   
      0.5 µl
      Bodicon Capping Enzyme (10 U/µl)   
      2 µl
      Total   
      20 µl
    4. Incubate at 37 °C for 2 h.
    5. Purify the RNA substrates by using phenol-chloroform extraction and ethanol precipitation methods.
  2. Prepare 10x MTase Buffer. The MTase buffer may be changed depending on different interested MTases.
  3. Combine the following reaction components in the order given for MTase assays:
    Purified MTases
    1 µg
    10x MTase Buffer
    3 μl
    GpppN-RNA or m7GpppN-RNA
    2 μg
    3 mM SAM
    0.5 μl
    [3H]-SAM (67.3 Ci/mmol, 0.5 μCi/μl)
    1 μl
    RNase inhibitor 40 U/μl
    1 μl
    RNase free water
    up to 30 μl
    Total
    30 μl
  4. Incubate the reaction at 30-37 °C depending on different MTases for 1.5 h.
  5. Transfer the tubes onto ice and add equal volume (30 μl) of 0.2% SDS, 20 mM EDTA.
  6. Keep the tubes on ice, add 1 ml of 10 mM NH4HCO3 (pH 8.5).
  7. Prepare 1 ml DEAE Sephadex column and equilibrated with 10 ml 10 mM NH4HCO3 (pH 8.5).
  8. Load the samples onto the equilibrated column.
  9. Wash the column with 10 ml of 10 mM NH4HCO3 (pH 8.5), 100 mM NaCl.
  10. Elute the samples with 1.5 ml of 10 mM NH4HCO3 (pH 8.5), 400 mM NaCl.
  11. Add equal volume scintillation liquid, mix well by vortexing and measure the signal using Liquid scintillation counter. The counting signal of [3H], which is transformed from [3H]-SAM to RNA substrates, represents the activity of tested MTases.

Recipes

  1. 10x MTase Buffer
    0.5 M Tris-HCl (pH 7.5 or 8.0)
    50 mM KCl
    20 mM MgCl2
    20 mM DTT
  2. Cap-0 cap structure (m7GpppN-RNA)
    Heat denatured RNA
    13.5 µl
    10x Bodicon Capping Buffer
    2 µl
    10 mM GTP
    2 µl
    3 mM SAM
    1 µl
    RNase inhibitor 40 U/µl
    0.5 µl
    Bodicon Capping Enzyme (10 U/µl)
    2 µl
    Total
    20 µl
  3. Non-methylated Cap-0 cap structure (GpppN-RNA)
    Heat denatured RNA
    13.5 µl
    10x Bodicon Capping Buffer
    2 µl
    10 mM GTP
    2 µl
    Inorganic pyrophospatase 0.1 U/µl
    1 µl
    RNase inhibitor 40 U/µl
    0.5 µl
    Bodicon Capping Enzyme (10 U/µl)
    2 µl
    Total
    20 µl
  4. MTase assay reaction mix
    Purified MTases
    1 µg
    10x MTase Buffer
    3 μl
    GpppN-RNA or m7GpppN-RNA
    2 μg
    3 mM SAM
    0.5 μl
    [3H]-SAM (67.3 Ci/mmol, 0.5 μCi/μl)
    1 μl
    RNase inhibitor 40 U/μl
    1 μl
    RNase free water up to
    30 μl
    Total
    30 μl

Acknowledgments

We thank Dr. Tero Ahola from University of Helsinki for the kindly help and advices for setting up this MTase assay. We thankfully acknowledge the University of Helsinki for providing research facilities and support during our visits. This work was supported by the China NSFC grants (81130083, 31170152 and 81271817).

References

  1. Chen, Y., Tao, J., Sun, Y., Wu, A., Su, C., Gao, G., Cai, H., Qiu, S., Wu, Y., Ahola, T. and Guo, D. (2013). Structure-function analysis of severe acute respiratory syndrome coronavirus RNA cap guanine-N7-methyltransferase. J Virol 87(11): 6296-6305.
  2. Chen, Y., Su, C., Ke, M., Jin, X., Xu, L., Zhang, Z., Wu, A., Sun, Y., Yang, Z., Tien, P., Ahola, T., Liang, Y., Liu, X. and Guo, D. (2011). Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex. PLoS Pathog 7(10): e1002294.
  3. Chen, Y., Cai, H., Pan, J., Xiang, N., Tien, P., Ahola, T. and Guo, D. (2009). Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase. Proc Natl Acad Sci U S A 106(9): 3484-3489.


How to cite this protocol: Chen, Y. and Guo, D. (2014). Biochemical Assays for MTase Activity. Bio-protocol 4(2): e1023. DOI: 10.21769/BioProtoc.1023; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook